Method of isolating human neuroepithelial precursor cells from human fetal tissue

ABSTRACT

A method for isolating human neuroepithelial precursor cells from human fetal tissue by culturing the human fetal cells in fibroblast growth factor and chick embryo extract and immunodepleting from the cultured human fetal cells any cells expressing A2B5, NG2 and eNCAM is provided. In addition, methods for transplanting these cells into an animal are provided. Animals models transplanted with these human neuroepithelial precursor cells and methods for monitoring survival, proliferation, differentiation and migration of the cells in the animal model via detection of human specific markers are also provided.

This patent application is a continuation of U.S. patent applicationSer. No. 13/435,424, filed Mar. 30, 2012, which is a continuation ofU.S. patent application Ser. No. 12/395,677, filed Mar. 1, 2009, nowissued as U.S. Pat. No. 8,168,174, which is a continuation of U.S.patent application Ser. No. 11/036,004, filed Jan. 14, 2005, now issuedas U.S. Pat. No. 7,517,521, which is a divisional of U.S. patentapplication Ser. No. 09/813,429, filed Mar. 21, 2001, now issued as U.S.Pat. No. 6,852,532, teachings of each of which are herein incorporatedby reference in their entireties.

BACKGROUND OF THE INVENTION

The demonstration that stem cells exist in the adult brain and spinalcord (Chiasson et al. J. Neurosci. 1999 19:4462-71Doetsch et al. Cell1999 97:703-16; Gage et al. J. Neurobiol. 1998 36:249-66; Johansson etal. Exp. Cell Res. 1999 253: 733-6; Kukekov et al. Exp. Neurol. 1999156:333-44; Pagano et al. Stem Cells 2000 18:295-300; Palmer et al. J.Neurosci. 1999 19:8487-97; Weiss et al. J. Neurosci. 1996 16:7599-609),that neurogenesis and gliogenesis are ongoing processes (Eriksson et al.Nat. Med. 1998 4:1313-7; Horner et al. J. Neurosci. 2000 20:2218-28;Johansson et al. Cell 1999 96:25-34; Kirschenbaum et al. Cereb. Cortex1994 4:576-89) and that stem cell populations can be modulated byextrinsic signals (Ahmed et al. J. Neurosci. 1995 15:5765-78;Forsberg-Nilsson et al. J. Neurosci. Res. 1998 53:521-30; Johe et al.Genes Dev. 1996 10:3219-40; Kalyani et al. Dev. Biol. 1997 186:202-23;Palmer et al. J. Neurosci. 1999 19:8487-97; Tsai, R. Y. and McKay, R. D.J. Neurosci 2000 20:3725-35; Vescovi et al. Exp. Neurol. 1999 156:71-83;Weiss et al. J. Neurosci. 1996 16:7599-609), has lead to a plethora ofpublications characterizing multipotent neural stem cells(NSCs)(Johansson et al. Exp. Cell Res. 1999 253:733-6; Kalyani et al.December Biol. 1997 186:202-23; Morrison et al. Cell 1999 96:737-49;Reynolds, B. A. and Weiss, S. Dev. Biol. 1996 175:1-13; Stemple, D. L.and Anderson, D. J. Cell 1992 71:973-85; Vescovi et al. Exp. Neurol.1999 156:71-83; Weiss et al. J. Neurosci. 1996 16:7599-609). What hasbecome clear is that several classes of multipotent cells exist, all ofwhich are nestin immunoreactive and capable of differentiating intoastrocytes, neurons and oligodendrocytes. Different populations of cellscan be distinguished by differences in culture conditions, self-renewalcapability, as well as in their ability to integrate and todifferentiate following transplantation (Gage, F. H. Science 2000287:1433-8; Rao, M. S. Anat. Rec. 1999 257:137-48).

Rodents NSCs isolated from different regions of the rostrocaudal axisand at different developmental stages exhibit differences indifferentiation potential, growth factor dependence and gene expression.For example, stem cells isolated at an early stage of embryogenesis fromthe developing spinal cord appear to require fibroblast growth factor(FGF) for survival, while stem cells isolated from more rostral portionsat later developmental stages seem equally responsive to FGF and/or toepidermal growth factor (EGF) (Reynolds, B. A. and Weiss, S. Dev. Biol.1996 175:1-13). Cells isolated from the ventricular zone express GFAP asa marker in the adult (Doetsch et al. Cell 1999 97:703-16) whilemultipotent cortical stem cells express polysialated NCAM (Marmur et al.Dev. Biol. 1998 204:577-91). Responses to neurotransmitters also appeardifferent. Ventricular zone stem cells proliferate in response toglutamate while subventricular zone stem cell turnover is reduced(Haydar et al. J. Neurosci 2000 20:5764-74). FGF-dependant stem cellscan generate EGF-dependent cells in vitro, suggesting that these cellsmay represent different developmental stages. The lineage relationshipbetween these various cells remains to be determined.

Less is known about human neural stem cells (hNSCs) isolated from fetaland adult tissue (Brannen, C. L. and Sugaya, K. Neuroreport 200011:1123-8; Carpenter et al. Exp. Neurol. 1999 158:265-78; Flax et al.Nat. Biotechnol. 1998 16:1033-9; Johansson et al. Exp. Cell Res. 1999253:733-6; Kukekov et al. Exp. Neurol. 1999 156:333-44; Pagano et al.Stem Cells 2000 18:295-300; Vescovi et al. J. Neurotrauma 199916:689-93; Vescovi et al. Exp. Neurol. 1999 156:71-83; Villa et al. Exp.Neurol. 2000 161:67-84). These cells give rise to glia and neurons, canbe grown under different culture conditions and show different growthfactor requirements. The lineage relationship among the variousidentified hNSCs and their relationship to previously described rodentstem cell populations remains to be determined. Indeed, comparativestudies of rodent and human-derived stem cells have been hampered by thelimited availability of cross-reactive reagents. For example, themonoclonal antibody nestin does not react with human cells and AC133, arecently identified stem cell marker, does not cross react withrat-derived NSC=s (Uchida et al. Keystone Symposium 2000).

Recently, hNSCs have become available through commercial sources such asClonetics (San Diego, Calif.) and Clonexpress (Gaithersberg, Md.). Firstpassage cells and growth conditions are available that generate neurons,astrocytes and possibly oligodendrocytes. Cells from Clonexpress have alimited differentiation capacity and generate cells that co-expressneuroglial markers (Piper et al. J. Neurophysiol. 2000 84:534-48).

Methods have now been developed for isolating and propagating atripotential human precursor cell which, upon characterization, has beenshown to share many features with a rodent-derived neuroepithelialprecursor (NEP)(Kalyani et al. Dev. Biol. 1997 186:202-231 Kalyani etal. J. Neurobiol. 1999 38:207-24). Like rodent-derived NEPs, hNEPs canbe grown as adherent cultures in FGF/chick embryo extract (CEE) and donot require leukemia inhibitory factor (LIF) or EGF for proliferationand survival. These cells can be induced to differentiate intoastrocytes, neurons and oligodendrocytes in culture. A subset of thehNEPs express AC133 and a small percentage are also GFAP positive.Further, hNEPs transplanted into the intact adult rat brain survive, canbe identified with human-specific antibodies, proliferate, differentiateinto neurons and glia and migrate extensively in a context dependentmanner.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a method for isolatingand propagating human neuroepithelial precursor cells from aheterogeneous population of human fetal cells. In the method of thepresent invention, commercially available human fetal cells are culturedwith fibroblast growth factor (FGF) and chick embryo extract (CEE).Cells expressing A2B5, NG2 and eNCAM are then immunodepleted to enrichfor a population of human neural precursor or stem cells.

Another object of the present invention is to provide a method fortransplanting into the central nervous system of an animal humanneuroepithelial precursor cells isolated via culturing of human fetalcells in FGF and CEE followed by immunodepletion of any cells expressingA2B5, NG2 and eNCAM from the culture.

Another object of the present invention is to provide nonhuman animalmodels for studying transplantation of human neural stem cells in thecentral nervous system. Nonhuman animal models of the present inventionare produced by transplanting into their central nervous systems humanneuroepithelial precursor cells isolated via culturing of human fetalcells in FGF and CEE followed by immunodepletion of any cells expressingA2B5, NG2 and eNCAM from the culture.

Yet another object of the present invention is to provide human specificmarkers and methods for monitoring survival, proliferation,differentiation and migration of human neuroepithelial precursor cellsin an animal model transplanted with human neuroepithelial precursorcells via detection of these human specification markers. For purposesof the present invention, human specific markers include human NCAM,GFAP, human nuclear antigen and human mitochondria.

DETAILED DESCRIPTION OF THE INVENTION

Human fetal tissue is commercially available from fetuses age 14 to 20weeks of age, a stage at which neurogenesis predominantly occurs. Atthis stage, neuron-restricted precursors are present and the number ofstem cells is significantly diminished. Thus, unlike neural tissueobtained from rodents prior to closure of the neural tube, only a smallsubset of cells in commercially available human fetal tissue age 14-20weeks are lineage negative cells with characteristics of humanneuroepithelial precursor cells. Accordingly, methods used to isolateneuroepithelial precursor cells in nonhuman animals are not directlyapplicable to isolating human neuroepithelial precursor cells fromcommercially available sources of human fetal tissue.

The present invention relates to a new method for isolating humanneuroepithelial precursor cells (hNEPs) from human fetal tissue age 14to 20 weeks. In this method, the small subset of precursor cells isselected via immunodepletion of differentiated cells and cultureconditions modified from those taught for rodent NEPs to be speciesspecific. Specifically, the human fetal cells are first grown inadherent culture in the presence of fibroblast growth factor (FGF) andchick embryo extract (CEE). Cells expressing A2B5, NG2 and eNCAM arethen depleted to enrich the population of multipotent precursor cells,approximately 50% of which stain for AC133/2.

Experiments were first performed to examine the heterogeneous populationof neural cells present in human fetal cells 14 to 20 weeks of age. Inthese experiments, lineage specific markers were used to analyze humanfetal cells from various stages of gestation. Cells were cultureddirectly onto fibronectin/laminin coated multiwell plates inmanufacturer=s media and fixed 24 hours after seeding. Results shown inTable 1 are from cells obtained at 19 weeks of gestation and arerepresentative of all ages tested.

TABLE 1 In vitro characterization of Human Fetal Cells Antibody HumanFetal Cells BIII tubulin 14.0% ± 1.4% GFAP 42.0% ± 3.8% AC-133/2 0%eNCAM 17.4% ± 2.1% NG2 0% A2B5 48.1% ± 2.9%As shown in Table 1, cultured fetal cells are a mixed populationcontaining a small percentage of BIII-tubulin positive neurons (14%). Anearly identical percentage of cells expressed eNCAM (17.4%) anddouble-labeling experiments indicated that these markers were primarilyco-expressed. A larger percentage of cells were GFAP immunoreactive(42%) and an equivalent percentage expressed A2B5 immunoreactivity. Noimmunoreactivity for NG-2, AC133/2, Ran-2, GD-3, 04 or Gal-C wasdetected. A small proportion of cells did not label with A2B5, GFAP orBIII-tubulin. These cells represented candidate precursor cells.

Several different culture conditions were then examined for theirability to enrich for the unlabeled precursor population. Significantenrichment was obtained when cells were grown in the presence of FGF andCEE. More specifically, cells were grown as adherent cultures onfibronectin/laminin coated dishes in DMEM/F12 medium containingadditives under similar conditions as described for rat cells byBottenstein and Sato (Proc. Natl Acad. Sci. USA 1979 76:514-7). It wasfound that the human cells required a relatively high concentration ofFGF for their survival. Preferred concentration of FGF demonstrated topromote survival range from about 10 to about 50 ng/ml. The addition ofCEE, preferably from about 5 to about 20% reduces the number ofdifferentiated cells present. Under these culture conditions asignificant percentage of cells began to express AC133/2immunoreactivity (53.2%). In addition, expression of NG-2 was detected.Table 2 provides a comparison of the fetal cell populations before andafter culturing in the presence of FGF and CEE.

TABLE 2 Comparison of In Vitro Characteristics of Human Fetal CellsHuman Fetal Cells cultured in FGF + Antibody Human Fetal Cells CEE BIIItubulin 14.0% ± 1.4% 20.7% ± 2.3% GFAP 42.0% ± 3.8% 59.2% ± 4.2%AC-133/2 0% 53.2% ± 6.0% eNCAM 17.4 ± 2.1 21.8% ± 2.2% NG2 0% 45.8% ±4.4% A2B5 48.1% ± 2.9% 51.6% ± 3.7%

Following culturing in FGF and CEE, the undifferentiated population waspurified by immunopanning and flow-activated cell sorting to removeeNCAM+, NG2+ and A2B5+ cells. Sorted and panned cells did not expresseNCAM, A2B5 or NG-2. A subset of the cells continued to express AC133/2and GFAP. It is believed that AC133/2 is expressed by a subset ofprecursor cells. This is consistent with recent reports that show thatAC133 can be used to isolate precursor cells (Corbeil et al. J. Biol.Chem. 2000 275:5512-20; Miraglia et al. Blood 1997 90:5013-21; Weigmannet al. Proc. Natl Acad. Sci. USA 1997 94:12425-30; Yin et al. Blood 199790:5002-12). It was found that of the 50% of human neural precursorcells exhibiting AC133/2 immunoreactivity, many differentiated intooligodendrocytes in culture. This is indicative of the possibility thatAC133 may recognize a more restricted precursor population, while a lessrestricted and more primitive precursor population may be AC133negative.

The cells are referred to herein as human neuroepithelial precursorcells or hNEPs based on their overall similarity to rodentneuroepithelial precursor cell cultures described by Kalyani et al.(Dev. Biol. 1997 186:202-23). The hNEPs of the present inventioncomprise a homogenous population of human precursor cells wherein 100%of the cells are immunoreactive with nestin, but are not immunoreactivewith NG-2, eNCAM or A2B5. Like rodent NEPS, hNEPs isolated via themethod of the present invention appear fibroblastic, divide rapidly inculture, and can be maintained in an undifferentiated state overmultiple passages. hNEPs can be induced to differentiate into neurons,astrocytes and oligodendrocytes using different culture conditions. Forexample, treatment with bFGF and NT3 resulted in a high percentage ofBIII-tubulin positive cells indicating the potential for neuronaldifferentiation. Cells cultured for 5 days in the presence of fetal calfserum stained positively for GFAP and presented a morphology consistentwith the presence of astrocytes in culture. The GFAP-positive (GFAP+)cells exhibited a variety of morphologies including bipolar and stellatecells. In addition, cells that were more well-spread displayed discreteGFAP+ filament bundles typical of mature astrocytes in culture. Cellstreated with bFGF for 2 days and then switched to a medium containingPDGF and T3 for days stained positively with antibodies against O4consistent with the presence of oligodendrocytes in culture.

Comparison of hNEPs isolated through the method of the present inventionwith other multipotent stem cells revealed a number of clear-cutdifferences. For example, unlike other previously described human stemcell populations, these cells do not require EGF for their survival andproliferation (Carpenter et al. Exp. Neurol. 1999 158:265-78; Fricker etal. J. Neurosci. 1999 19:5990-6005; Quinn et al. J. Neurosci. Res. 199957:590-602; Reynolds, B. A. and Weiss, S. Dev. Biol. 1996 175:1-13;Villa et al. Exp. Neurol. 2000 161:67-84). Further, unlike the cellsdescribed by Carpenter et al. (Exp. Neurol. 1999 158:265-78), thesecells do not require LIF for their proliferation. hNEPs also readilygrow as adherent cultures and can be passaged using EDTA alone. Only asmall amount of cell death is observed when cells are passaged in thisway compared to the large amount of cell death observed when neurospheretype cultures are treated similarly (Svendson et al. J. Neurosci.Methods 1998 85:141-52).

The hNEPs isolated via the method of the present invention have beendemonstrated to act as multipotent precursor cells capable ofdifferentiating into neurons, astrocytes and, to a limited extent,oligodendrocytes, not only in vitro but also following transplantationinto the intact adult rat subventricular zone and striatum.

In in vitro experiments, growth factor withdrawal promoteddifferentiation of hNEPs. Neuronal markers appeared early followed byastrocytic and oligodendrocytic markers. Neurons constitutedapproximately 20% of the cells. The remaining cells appearedundifferentiated even under differentiation conditions, i.e. removal ofFGF and CEE and addition of retinoic acid. Despite exposing the cells toseveral well established tests for inducing differentiation, no bias ofdifferentiation towards any particular lineage other than astrocytescould be achieved. Serum exposure converted virtually the entirepopulation to GFAP positive cells. Promotion of myelination in culturewas also not achieved. Oligodendrocytes as identified by O4 or Gal-C didnot appear to survive for prolonged periods and myelin markers such asMBP and PLP were undetectable even after co-culture with rat neurons.Thus, it is believed that human oligodendrocyte precursors requirespecific signals for maturation that were absent in the cultureconditions used.

The ability to differentiate hNEPs and rat NEP cells in culturepermitted testing of markers that are expressed at different stages ofdevelopment and comparison of their expression on rat versus humancells. Several markers of immense value in transplant experiments wereidentified via this comparison. For example, it was found that humanspecific NCAM, GFAP, human nuclear antigen and human mitochondria can beused to follow transplanted human cell populations. Furthermore, humanneural cell adhesion molecule (hNCAM) and human glial fibrillary acidicprotein (hGFAP) are extremely useful in providing a dual label that notonly identified the cell as human but also identified its phenotype.Table 3 provides a summary of markers useful in identifying, monitoringand determining the phenotype of human cells following transplantationof hNEPs in animal models. Each antibody was tested on cultures of ratCNS cells to assess cross-reactivity issues. Results are summarized inTable 3.

TABLE 3 Immunohistochemical Characterization of Human NeuroepithelialPrecursors Rat Cross In In Reactivity Antibody Specificity Vitro Vivo InVitro BrdU Dividing Cells +/− +/− + AC-133/2 Hu stem cells/ +/− +/− −progenitors hMito Hu mitochondria + + − MAB 1281 Hu nuclei + + − HNCAMHu neural cell − +/− − adhesion molecule BIII Rat and Hu neurons − +/− +tubulin HGFAP Hu glial fibrillary +/− +/− − acidic protein GFAP Rat andHu GFAP +/− +/− + MAB 328 Rat and Hu − +/− + oligos/myelin Note: +/−gaining refers to staining that does not label all human cells.

Accordingly, also provided in the present invention are methods formonitoring survival, proliferation, differentiation and migration ofhuman cells following transplantation of hNEPs into animal models. Forexample, survival of hNEPs transplanted into the adult rat brain andtheir identification can be performed via species-specific antibodiessuch as antibodies specific for human specific NCAM, GFAP, human nuclearantigen or human mitochondria. The localization of transplanted hNEPs isfirst ascertained at various days post-transplantation via BrdU stainingwhich identifies pre-labeled hNEPs. For example, at 2 and 7 dayspost-transplantation, in vitro staining of samples of transplanted cellsshowed approximately 70% BrdU labeling efficiency. Human-specificmarkers, as described in Table 3, are then utilized to locate andidentify transplanted hNEPs. The MAB 1281, a marker specific for humannuclei (hNuc), and hMito, a marker for human mitochondria, can be usedto identify transplanted hNEPs along the injection tract and in adjacenthost tissue. Several other markers can be used to identifysubpopulations of the transplanted hNEPs and to reflect the capacity ofthese cells to retain a progenitor status, as well as to differentiateinto neurons and glia. Anti-AC133/2, shown, supra, to label asubpopulation of the transplanted hNEPs, can be used to determinewhether any hNEPs retain AC133/2 immunoreactivity in situ. At 2 and 7days post-transplantation, the pattern of staining for AC133/2 positivecells coincides with DAPI nuclear labeling. Human NCAM staining isuseful in identifying a subset of those transplanted hNEPs that havedifferentiated along the neuronal-restricted lineage. Human-specificGFAP antibody can be used to identify human astrocytes in situ. Inaddition, distinctive perikaryal staining can be used to identify cellbodies and process labeling can be used to identify long processes withmultiple varicosities.

Further provided in this invention are methods for using hNEP cellsisolated in accordance with the method of the present invention fortransplantation into the central nervous system of animals, including,but not limited to, experimental animal models as well as humans. Untilnow, human to animal model transplantation studies have been hampered bythe limited availability of cross-reactive reagents. However, theability of isolate hNEPs via the method of the present invention hasenabled identification of several markers specific to human cellsincluding antibodies to human nuclei, human mitochondria, human-specificNCAM and human-specific GFAP.

Using both lineage-specific and human-specific markers, transplantedhNEPs isolated in accordance with the method of the present inventionwere demonstrated to survive transplantation in the intact adult ratbrain and to have the capacity to generate neurons, astrocytes and alimited number of oligodendrocytes. In the case of hNEP-derivedastrocytes, differentiation appeared to be regionally specific. Thecontinued presence of undifferentiated transplanted human hNEPs,identified by the lack of more mature lineage-restricted markers,suggests that early precursors are still present at 28 days followingtransplantation and are associated with proliferative areas of theintact adult rat brain. In all, undifferentiated and more differentiatedhNEP cell derivatives appeared to integrate in a non-disruptive mannerinto many types of adult brain tissue.

hNEPs, or their progeny, were also found to continue to divide aftertransplantation. However, no evidence of uncontrolled proliferationincluding masses, heterotopias or tumors were observed. In fact, eventhe initial injection tract contained only a small number of cells onemonth after transplantation indicating that cells either migrated awayor responded to endogenous signals.

hNEPs were also found to migrate quite extensively, but not randomly.Cells preferentially followed three paths when transplanted into theSVZa: an anterior stream towards the olfactory bulb; a directed streamof cells to the ventricular zone; and a posterior stream along thecorpus callosum towards the occipital cortex. Little migration into thehippocampus, cerebellum or cortex was seen. Transplants into thestriatum showed much more limited migration, which was primarilyastrocytic and involved migration through the parenchyma. Overall theseresults indicate that hNEPs and their derivatives follow endogenous cuesor permissive paths. The ability to travel down the RMS suggests thathNEPs or their derivatives can follow non-radial glial pathways ofmigration.

In addition, mature neurons were observed after only a month followingtransplantation. These cells clearly matured during this time period,extended processes and appeared to integrate, indicating thattransplanting cells into immunosuppressed intact rat brain may be auseful model for studying the developmental biology of human precursorcells, as well as for evaluation of the many potential therapeutic usesof these cells.

Finally, while differentiation of hNEPS into oligodendrocytes wasminimal, oligodendrocytes were observed along the injection tract,presumably because of local damage caused by the injection. Thus, it isbelieved that hNEPs will like readily myelinate in an appropriate modeland have begun experiments to test this possibility.

All of the transplantation studies were performed with freshly pannedand sorted hNEPs. BrdU pre-labeling, at approximately 70% labelingefficiency, was used to identify transplanted cells at 2 and 7 days.Other animals grafted with unlabeled cells were examined at 28 daysusing human specific markers. Transplanted BrdU-positive cells wereobserved in all animals at 2 and 7 days post-transplantation in allgraft sites and in the respective injection tracts. In addition,transplanted cells could be identified using human specific antibodiesdirected against hNCAM, hGFAP, hMito and hNuc. All transplanted animals(n=36) exhibited evidence of differentiation that varied with the siteof transplantation and the time of sacrifice.

Transplanted human NEP cells differentiated into neurons and gliafollowing transplantation into intact adult SVZa and striatum. Graftedcells were examined with both cell type and human specific markersfollowing transplantation (Table 3). Soon after grafting (2 days), largenumbers of BrdU-labeled cells were observed at both sites. A smallnumber of BrdU-labeled cells stained for GFAP, however, the majority ofcells did not react with any of the markers listed in Table 3 indicatingthat many of the grafted cells remained undifferentiated 48 hoursfollowing implantation. Human specific GFAP-labeled (hGFAP+) cellsexhibited a unipolar morphology irrespective of the site oftransplantation. The hGFAP-labeled processes appeared much shorter andthicker than any of the endogenous populations of astrocytes asdetermined by a comparative analysis of the staining pattern obtainedwith non-species specific polyclonal antisera to GFAP. These resultsindicate that 48 hours after transplantation, the majority of cellsremained undifferentiated, while a small portion had differentiated intoastrocytes, but none into mature neurons or oligodendrocytes.

At later time points (7 and 28 days), larger numbers of grafted cellsstained for neuron-specific and astrocyte-specific markers, while only asmall subset of cells labeled for oligodendrocyte markers. Humanspecific NCAM and hNuc/BIII tubulin-double positive cells and theirprocesses were observed at both graft sites, in the adjacent parenchymaand in the white matter, suggesting that many of the cellsdifferentiated into neurons. These cells possessed unipolar, bipolar andmulti-polar processes that in some cases extended hundreds of micronsinto the cortex, along white matter tracts and into the RMS. Acomparison of hNCAM and hNuc/BIII tubulin+ cells at 2, 7 and 28 daysindicated that both the number of cell bodies as well as the length andcomplexity of their processes increased over time, suggesting that cellgrowth/hypertrophy followed differentiation and that this processincreased in incidence over time. Human specific GFAP-positive cellswere observed at both graft sites, throughout the ipsilateral corpuscallosum extending to the occipital cortex and ipsilateral striatum. Atleast three distinct phenotypes were apparent, which appeared to beregionally specific. HGFAP-labeled cells in the striatum had a stellatemorphology similar to those of the endogenous population, many of whichappeared to contact blood vessels. HGFAP-positive cells located alongthe length of the callosum had long processes similar to the endogenouspopulation of white matter astrocytes, while hGFAP labeled cells locatedat the wall of the lateral ventricle had processes which extended towardthe ependyma in a manner similar to the endogenous population, but withprocesses that were much thicker. While a significant subset of cellslabeled for neuron and astrocyte markers, only a few cells expressedmarkers consistent with an oligodendrocyte phenotype. When MAB 328antibody coupled with antisera to human nuclear antigens were used todetect human specific oligodendrocyte differentiation, MAB328/hNuc-double labeled cells were observed only where the injectiontract penetrated the corpus callosum. Differentiation intooligodendrocytes is believed to be induced by damage from thepenetrating stab wound through the white matter. However, the smallnumber of MAB 328/hNuc-double labeled cells indicated that a verylimited amount of oligodendrocyte differentiation had occurred.

Not all of the transplanted cells labeled with lineage-restrictedmarkers. At 2 and 7 days, significant numbers of BrdU-labeled cells,which double labeled with antisera against human mitochondrial antigens(hMito), did not double label with other markers listed in Table 3.Thus, many cells remained undifferentiated. These cells were present atall grafts sites, injection tracts, as well as in the ipsilateralsubependymal zone, corpus callosum, and in the ipsilateral RMS.Similarly, at 28 days, a significant number of hNuc-labeled cells didnot co-label with other cell type specific markers. However, at day 28,larger numbers of these cells were observed along the wall of theipsilateral lateral ventricle, in the ipsilateral RMS, and in the corpuscallosum extending, in some cases, toward the occipital cortex. Thepresence of hNEP cells in the subependymal zone of the ipsilaterallateral ventricle and in the RMS at 28 days indicates that a subset ofthe grafted cells had integrated and continued to proliferate in amanner similar to the endogenous population of precursor cells.

To assess whether hNEP cells continued to proliferate followingtransplantation, two separate experiments were performed. BrdU-labeledcells were implanted and analyzed following transplantation (N=19).Several pieces of evidence indicate that transplanted hNEP cellscontinue to proliferate following transplantation into intact adult SVZaand striatum. At both graft sites, the number of BrdU-labeled cellsdecreased with time from transplantation, while the number anddistribution of cells labeled with human-specific antisera increased. Inthe latter case many hNuc-positive cells remained undifferentiated asdetermined by a lack of staining with cell type specific orlineage-restricted markers, thus indicating that early precursorscontinued to be generated over time. At all time points at both graftssites, intensely labeled BrdU-positive cells were surrounded by lessintensely labeled BrdU+ cells that were often distributed at theperiphery of the grafted cell mass.

To further assess the potential of hNEPs to divide in situ, BrdU wasinjected i.p. one day prior to the sacrifice of 28 day old grafts (N=8)and compared with BrdU distribution in non-transplanted, BrdU-injectedcontrol animals (N=3). At 28 days, BrdU-positive cells were identifiednear the injection site and along the injection tracts in areas where noBrdU-labeled cells were observed in non-transplanted control animalsindicating that cell proliferation continued at the graft site. Lastly,a large number of hNuc-labeled cells that did not co-label with moremature lineage-restricted markers were observed in the subventricularzone suggesting that these cells had integrated and continued to dividein an effort to function as those previously identified endogenousneural stem cells of the SVZa (Doetsch et al. Cell 1999 97:703-16;Doetsch et al. J. Neurosci. 1997 17:5046-61; Garcia-Verdugo et al. J.Neurobiol. 1998 36:234-48; Goldman et al. J. Neeurooncol. 1995 24:61-4;Lois, C. and Alvarez-Buyll, A. Proc. Natl Acad. Sci. USA 199390:2074-7).

Migration of hNEP cells following transplantation in the intact adultrat brain was region-specific. The distribution of BrdU-labeled cells,as well as those identified with human-specific antisera, indicated thatgrafted human NEP cells migrated extensively along the ipsilateralrostral-caudal axis. In addition, the distribution of cells over timewas graft-site dependent. In SVZa grafted animals at 2 days, intenselyBrdU-labeled cells were restricted to the anterior subventricular zonebelow the corpus callosum within the injection tract, the corpuscallosum, and extended medially to surround the rostral and lateralaspect of the lateral ventricle. The nuclei of cells in the corpuscallosum were elliptical in shape and oriented along the white matter.At 7 days, hNEPs were observed in the ipsilateral rostral migratorystream (RMS) extending into the olfactory bulb. These cells displayed anovoid-shaped nucleus and were aligned along the rostral-caudal axis ofthe RMS, suggesting rostral migration with features consistent with thebehavior of endogenous cells. At 28 days, large numbers of hNEPs labeledwith antisera against BrdU, hNuc and hMito were found throughout thesubventricular zone and all levels of the RMS extending into theipsilateral olfactory bulb. Many of the cells located in the RMSexhibited a bipolar morphology and labeled with antisera against BIIItubulin and human-specific NCAM. In addition, transplanted cellsidentified with hGFAP immunoreactivity were dispersed along therostral-caudal axis of the corpus callosum, indicating that cells alsohad migrated medially and laterally along the white matter, in somecases extending caudally to the occipital cortex. HGFAP-positive cellsin white matter exhibited extensive processes similar to endogenouswhite matter astrocytes.

Cells grafted into the striatum displayed a different pattern ofmigration. At both 2 and 7 days, the majority of grafted cells wererestricted to the graft site as a deposit of cells that stainedpositively for hNuc/BIII, hNCAM and hGFAP. Additionally, unlike thosecells transplanted in the SVZa, the nuclei of these cells did notexhibit an elliptical morphology. By 28 days, however, transplantedhNEPs were found distributed throughout the ipsilateral striatum, alongthe more caudal aspects of the corpus callosum, the caudal subependymalzone of the lateral ventricle and posteriorly toward the occipitalcortex. The transplanted cells appeared to be migrating in a radialpattern about the site of implantation. The majority of cells were hGFAPpositive. In addition, hNEPs could also be found migrating posteriorlyalong the border of the fimbria and the caudal striatum near the wall ofthe lateral ventricle. Similar to the RMS, many undifferentiated BrdU+cells were found. These cells were present in a chain-like fashion withelliptical nuclei oriented in the rostral-caudal axis. Humanmitochondrial staining further revealed that these cells hadmitochondria-rich processes aligned along the plane of the ventralhippocampal commissure, indicating that these transplanted hNEPs weremigrating to the hippocampus. A small number of transplanted BrdU+ cellswere also found incorporating into the ependymal lining of the posteriorregion of the lateral ventricle.

Thus, as demonstrated herein, the hNEPs isolated in accordance with themethod of the present invention are useful in developing nonhuman animalmodels for the study of transplantation of these human cells into thecentral nervous system. For purposes of the present invention, bynonhuman animal it is meant any animal with a central nervous systemcomprising a brain and spinal cord. In a preferred embodiment, theanimal model comprises a rodent, such as a rat or mouse, a lagomorph, acanine or a primate. However, as will be understood by those of skill inthe art upon reading this disclosure, other animals identified as usefulin the study of central nervous system cell transplantations can also beused. In the animal models of the present invention, hNEPs isolated fromhuman fetal tissue in accordance with the method described herein aretransplanted into either the brain or spinal cord of the animal. Thetransplanted hNEPS can then be monitored for survival, proliferation,differentiation, and migration via the methods and markers describedherein.

Expansion and purification of a human-derived multipotent precursorderived from a commercially available source of fetal tissue will alsofacilitate the development of cell-based therapies for the restorationof CNS tissue function in humans. As demonstrated herein, hNEP cellsisolated via the method of the present invention survive, proliferate,migrate and differentiate in vivo in an animal model fortransplantation. Based upon these animal experiments, it is expectedthat the cells will function similarly when transplanted into thecentral nervous system of humans. Accordingly, the hNEP cells isolatedin accordance of the method of the present invention are expected touseful in conditions wherein replacement of neural cells is needed.Examples of such conditions include, but are not limited to, Parkinson=sdisease, Huntington=s disease, stroke and epilepsy. Several clinicaltrials with other neural cell types are currently being conducted forthese diseases. Similar protocols and procedures used in these clinicaltrials with other neural cells can be adapted routinely by those ofskill in the art for use with the hNEPs of the present invention.

The following nonlimiting examples are provided to further illustratethe present invention.

EXAMPLES Example 1 Culture of Human Neural Stem Cells

Human neural progenitor cells derived from fetal tissue were acquiredfrom Clonetics (San Diego, Calif.). Frozen aliquots of cells were thawedand plated on fibronectin/laminin-coated multiwell dishes in NeuralProgenitor Cell Basal Medium (NPBM, Clonetics) supplemented with humanrecombinant basic fibroblast growth factor, human recombinant epidermalgrowth factor, neural survival factors, 5 mg/ml gentamicin and 5 μg/mlamphotericin-B (Singlequots, Clonetics). Cultures were incubated at37^(∀)C, 5% CO₂ and fixed 24 hours later. These wells were subsequentlyprocessed for immunocytochemistry to assess the starting population ofClonetics cells. In parallel, Clonetics cells were thawed andimmediately plated on fibronectin/laminin-coated flasks (Greiner) andcultured in Neuroepithelial Precursor (NEP) medium that consisted ofDMEM-F12 (Life Technologies, Grand Island, N.Y.) supplemented withadditives as described by Bottenstein and Sate (Proc. Natl Acad. Sci.USA 1979 76:514-7), basic fibroblast growth factor (bFGF, 10 ng/ml,Peprotech, Rocky Hill, N.J.) and chick embryo extract (CEE, 10%)prepared as described by Stemple and Anderson (Cell 1992 71:973-85).Unattached cells typically formed floating spheres. After 24 hours inculture, spheres were removed, gently triturated and re-combined withthe attached cells. NEP media was exchanged every other day.

Example 2 Isolation of Human Neuroepithelial Precursor Cells (hNEPs)

After 5 days in culture, immunopanning and flow-activated cell sortingwere used to remove eNCAM⁺, NG2⁺, and A₂B₅ ⁺ cells. Briefly, cells weretreated with 5 mM EDTA (Life Technologies) and the suspension plated onan eNCAM antibody (5A5, Developmental Studies Hybridoma Bank)-coateddish to allow binding of all eNCAM⁺ cells to the plate. ENCAMantibody-coated dishes were prepared by sequentially coating tissueculture dishes with an unlabeled anti-mouse IgM antibody (10 μg/ml)overnight, rinsing dishes with DPBS, followed by coating with 5A5hybridoma supernatant for 1-3 hours at 37^(∀)C. Plates were washed twicewith DPBS prior to plating neural progenitor cells. After a 30 minuteexposure period, unbound cells (eNCAM cells) were removed and platedonto a dish coated with antibodies to NG2 for 30 minutes. NG2 panningdishes were made by coating dishes with an NG2 antibody (1:50) for 1-3hours at 37^(∀)C. The supernatant was then removed (eNCAM/NG2 cells) andimmunostained for A₂B₅. Cells were exposed to antibodies to A₂B₅ (1:2,Developmental Studies Hybridoma Bank) in NEP media for 1 hour to stainthe membranes of live A₂B₅ ⁺ cells. All cells were then sent through aflow-activated cell sorter to remove the population of A₂B₅ ⁺ cells.After sorting, the negative population (human NEPs) was propagated inNEP media on fibronectin/laminin coated T-75 flasks prior totransplantation studies. NEP media was exchanged every other day.

Example 3 Generation of Neurons, Oligodendrocytes and Astrocytes fromhNEPs

Panned/sorted populations of human NEPs were plated onfibronectin/laminin-coated 12 mm coverslips in various conditions topromote differentiation. Coverslips were fixed with 4% paraformaldehydeat the times established below. To induce neuronal differentiation,cells were exposed to bFGF 10 ng/ml) and NT3 (10 ng/ml, Peprotech).After 5 days in culture, fixed cultures were stained using antibodies to(β-III tubulin to assess the capacity of these cells to differentiateinto neurons. For oligodendrocyte differentiation, cells were plated ina bFGF (10 ng/ml)-containing medium for 2 days and then were switched toa medium containing PDGF (10 mg/ml, Upstate Biotech, Waltham, Mass.) andT3 (50 nM, Sigma Chemical Co. St. Louis, Mo.) for 7 days. Antibodies toO4 were used to identify oligodendrocytes in culture. For astrocyticdifferentiation, cells were cultured for 5 days in the presence of fetalcalf serum (10%, Life Technologies). Fixed cultures were stained withantibodies to GFAP to identify mature astrocytes.

Example 4 Immunocytochemistry

Cultures were stained using antibodies against A₂B₅ (1:2, DevelopmentalStudies Hybridoma Bank), AC133/2 (1:100, Miltenyi Biotec, Auburn,Calif.), (β-III tubulin (1:1000, Sigma), eNCAM (1:2, 5A5, DevelopmentalStudies Hybridoma Bank), GFAP (1:2000, Dako, Carpinteria, Calif.), NG2(1:50), and O4 (1:2, BMB hybridoma). Following fixation, cultures weretreated with 0.5% Triton (Triton X-100, Sigma) in PBS for 2 minutes toaccess intracellular antigens. Fixed coverslips or plates were thentreated with primary antibodies in a blocking solution containing Hank=sbalanced salt solution and 5% calf serum for 1 hour at room temperature.Following 3 washes with PBS, cultures were incubated in the appropriatesecondaries (1:220) conjugated to either Texas Red or Alexa 488(Molecular Probes, Eugene, Oreg.) for 1 hour at room temperature.AC133/2 staining required amplification with a biotinylated secondary,followed by a streptavidin-alexa 488 conjugated tertiary. All cultureswere counterstained with DAPI (Molecular Probes) to identify cellnuclei.

Example 5 Cell Preparation for Transplantation

One day prior to transplantation, cultured human NEP cells were treatedwith 10 ng/ml bromo-deoxyuridine (BrdU, Sigma) in culture mediumovernight at 37EC. The following day, media was removed and the cellswere exposed to 0.25% trypsin containing 1 mM EDTA (Life Technologies)for 8 minutes at 37EC. Following trypsin treatment, cells werecentrifuged for 3 minutes at 1000 rpm. The pellet was then resuspendedin L15 media (Life Technologies) containing 0.04% DNase (WorthingtonBiochemical Corp., Freehold, N.J.). The suspension was triturated with afire-polished glass pipette and the cells counted with a hemacytometer.The cells were centrifuged one final time to remove DNase and thenresuspended in L15 at 20,000 cells/μl in preparation fortransplantation.

Example 6 Immunosuppression and Animal Surgery

Rats were treated with daily injections of cyclosporin (Novartis, EastHanover, N.J.) (10 mg/kg) i.p. beginning the day before surgery andending at time of sacrifice. All animals sacrificed at 2 and 7 daysreceived transplants of BrdU labeled cells, while those sacrificed at 28days received unlabeled cells and two injections of BrdU (100 mg/kg,spaced 2 hours apart) one day prior to sacrifice. Non-transplantedanimals served as controls with some animals receiving BrdU injectionsone day prior to sacrifice (N=3). Sterile surgical procedures and animalcare were conducted according to IACUC approved guidelines. Adult(250-300 g) male Fisher 344 rats were anesthetized with a mixture ofketamine 65 mg/kg), xylazine (7.5 mg/kg), acepromazine (0.5 mg/kg) andplaced in a stereotaxic frame (Stoelting, Wood Dale, Ill.). The nose barwas set 2.7 mm below the intra aural line. A midline incision was madeand a 500 μm hole was created in the skull using a stainless steeldental drill. Following careful exposure of the underlying cortex, a 320μm diameter needle was lowered into the brain. All animals received a 3μl injection (20,000 cells/μl) of human NEPs diluted in L15 mediainjected at 1 μl/minute in the anterior subventricular zone (SVZa)(N=22)at +1.4 mm bregma, +1.5 mm lateral, −5.0 mm deep from the brain surface,or the caudal striatum (N=14) at −1.4 mm bregma, +4.3 lateral, −5.0 mmdeep from brain surface. The needle was left in the brain for 4 minutesto allow for equilibration and then slowly withdrawn. The overlyingscalp was then closed with 5/0 silk suture. All animals had access tofood and water ad libitum pre- and post-surgery.

Example 7 Histological Procedures

At 2, 7 and 28 days post-transplantation, rats were deeply anesthetizedand perfused transcardially with 250 ml of phosphate-buffered saline(PBS, pH 7.4) followed by 250 ml of fresh 4% paraformaldehyde at a rateof 50 ml/minute. The brain was removed and post-fixed overnight in 4%paraformaldehyde at 4EC. The specimens were transferred toneutral-buffered PBS and stored at 4EC. Fifty micron sagittal andhorizontal tissue sections were cut in cold 1×PBS using a vibratome(Ted-Pella Inc., Redding, Calif.).

Example 8 Immunostaining

Vibratome sections were permeabilized with 0.5% Triton X-100 (Sigma) inPBS for 30 minutes and immuno-blocked using either a dilute goat (4%) orhorse serum (3%) in PBS containing 0.3% triton. Sections stained forBrdU were incubated in ice-cold 0.1N HCl for 10 minutes and then 2.0NHCl for 30 minutes at 37EC. In general, sections were incubated inprimary antibody diluted in blocking solution overnight (18-20 hours) at4EC. The following primary antibodies were used: monoclonal ratanti-BrdU (1:150; Accurate Chemicals, Westbury, N.Y.); monoclonalhuman-specific anti-mitochondria (anti-hMito; 1:500; Chemicon, Temecula,Calif.); monoclonal human specific against nuclear antigens (anti-hNuc;1:200; Chemicon); monoclonal against neuronal nuclear antigens (NeuN;1:220; Chemicon); monoclonal human-specific neural cell adhesionmolecule (anti-hNCAM; 1:500; Chemicon); polyclonal antibodies againstglial fibrillary acidic protein (GFAP; 1:2000; Dako Corp., Carpinteria,Calif.); monoclonal antibody directed against human GFAP (1:1000;Sternberger Monoclonals, Lutherville, Md.) and monoclonal mouseanti-oligodendrocytes/myelin (MAB 328; 1:1000; Chemicon). Next, thesections were rinsed in PBS and incubated in alexa 488 or alexa594-conjugated goat anti-mouse IgG1 (1:220 in PBS+0.3% triton) for 1hour at room temperature to visualize anti-hNuc and anti-NeuN.Alexa-conjugated goat anti-rabbit IgG (Molecular Probes, Eugene, Oreg.)was used to visualize anti-GFAP. Similarly, alexa-conjugated goatanti-mouse IgM was used for MAB 328 staining. Biotinylated secondarieswere used to amplify all other reactions at a concentration of 1:220 for1 hour at room temperature diluted in PBS+0.3% triton. For anti-BrdU,goat anti-rat IgG; anti-hMito, goat anti-mouse IgG1; anti-hNCAM, goatanti-mouse IgG1; anti-hGFAP, goat anti-mouse IgG1 (all biotinylatedreagents purchased from Southern Biotech. Assoc., Birmingham, Ala.).Following secondary treatment, a streptavidin-conjugated tertiary wasapplied to the sections for 50 minutes at room temperature to visualizethe reaction. All tertiaries were conjugated to either Texas Red orAlexa 488 (Molecular Probes) and also used as a concentration of 1:220in PBS. All sections were counterstained with DAPI to identify cellnuclei.

Stained sections were visualized using the appropriate fluorescentfilters on a Nikon E600 upright epifluorescent microscope.Representative images were captured using a digital color video camera(CoolSnap, RS Photometrics). For double and triple-stained samples, eachimage was taken using only one emission filter at t time. Layeredmontages were then prepared using Adobe Photoshop software on a PCcomputer.

What is claimed is:
 1. A method for transplanting an isolated populationof human neuroepithelial precursor cells into an animal comprising: (a)isolating human neuroepithelial precursor cells in accordance with amethod comprising depleting from human fetal neural tissue any cellsexpressing A2B5, NG2 and eNCAM so that an isolated population of humanneuroepithelial precursor cells remains; and (b) transplanting theisolated human neuroepithelial precursor cells into the animal.